Carey Krajewski

Nuclear gene sequences provide evidence for the monophyly of australidelphian marsupials

Relationships among the seven extant orders of marsupials remain poorly understood. Most classifications recognize a fundamental split between Ameridelphia, which contains the American orders Didelphimorphia and Paucituberculata, and Australidelphia, which contains four Australasian orders (Dasyuromorphia, Diprotodontia, Notoryctemorphia, and Peramelina) and the South American order Microbiotheria, represented by Dromiciops gliroides. Ameridelphia and Australidelphia are each supported by key morphological characters with dichotomous character states. To date, molecular studies indexing all marsupial orders have reported inconclusive results. However, several studies have suggested that Dromiciops is nested within Australidelphia. This result has important implications for understanding the biogeographic history of living marsupials. To address questions in higher-level marsupial systematics, we sequenced portions of five nuclear genes (Apolipoprotein B gene; Breast and Ovarian cancer susceptibility gene 1; Recombination activating gene 1; Interphotoreceptor retinoid binding protein gene; and von Willebrand factor gene) for representatives of all orders of marsupials, as well as placental outgroups. The resulting 6.4kb concatenation was analyzed using maximum parsimony, distance methods, maximum likelihood, and Bayesian methods. tests were used to examine a priori hypotheses. All analyses provided robust support for the monophyly of Australidelphia (bootstrap support=99-100%; posterior probability=1.00). Ameridelphia received much lower support, although this clade was not rejected in statistical tests. Within Diprotodontia, both Vombatiformes and Phalangeriformes were supported at the 100% bootstrap level and with posterior probabilities of 1.00.

The origin of the Australasian marsupial fauna and the phylogenetic affinities of the enigmatic monito del monte and marsupial mole

Alternative hypotheses in higher–level marsupial systematics have different implications for marsupial origins, character evolution, and biogeography. Resolving the position of the South American monito del monte (Order Microbiotheria) is of particular importance in that alternate hypotheses posit sister-group relationships between microbiotheres and taxa with disparate temporal and geographic distributions: pediomyids; didelphids; dasyuromorphians; diprotodontians; all other australidelphians; and all other marsupials. Among Australasian marsupials, the placement of bandicoots is critical; competing views associate bandicoots with particular Australasian taxa (diprotodontians, dasyuromorphians) or outside of a clade that includes all other Australasian forms and microbiotheres. Affinities of the marsupial mole are also unclear. The mole is placed in its own order (Notoryctemorphia) and sister–group relationships have been postulated between it and each of the other Australasian orders. We investigated relationships among marsupial orders by using a data set that included mitochondrial and nuclear genes. Phylogenetic analyses provide support for the association of microbiotheres with Australasian marsupials and an association of the marsupial mole with dasyuromorphs. Statistical tests reject the association of diprotodontians and bandicoots together as well as the monophyly of Australasian marsupials. The origin of the paraphyletic Australasian marsupial fauna may be accounted for by (i) multiple entries of australidelphians into Australia or (ii) bidirectional dispersal of australidelphians between Antarctica and Australia.

PHYLOGENY OF CRANES (GRUIFORMES: GRUIDAE) BASED ON CYTOCHROME-B DNA SEQUENCES

ABS?RACr.--DNA sequences spanning 1,042 nucleotide bases of the mitochondrial cytochrome-b gene are reported for all 15 species and selected subspecies of cranes and an outgroup, the Limpkin (Aramus guarauna). Levels of sequence divergence coincide approximately with current taxonomic ranks at the subspecies, species, and subfamilial level, but not at the generic level within Gruinae. In particular, the two putative species of Balearica (B. pavonina and B. regulorum) are as distinct as most pairs of gruine species. Phylogenetic analysis of the sequences produced results that are strikingly congruent with previous DNADNA hybridization and behavior studies. Among gruine cranes, five major lineages are identified. Two of these comprise single species (Grus leucogeranus, G. canadensis), while the others are species groups: Anthropoides and Bugeranus; G. antigone, G. rubicunda, and G. vipio; and G. grus, G. monachus, G. nigricollis, G. americana, and G. japonensis. Within the latter group, G. monachus and G. nigricollis are sister species, and G. japonensis appears to be the sister group to the other four species. The data provide no resolution of branching order for major groups, but suggest a rapid evolutionary diversification of these lineages. Received 19 March 1993, accepted 19 August 1993. THE 15 EXTANT SPECIES of cranes comprise the nominate family (Gruidae) of the order Gruiformes, and are currently divided into two subfamilies, Balearicinae and Gruinae (Brodkorb 1967). Balearicine cranes are anatomically unspecialized relative to gruines and are represented by only two extant species in the genus Balearica (the crowned cranes of Africa). Gruines share derived anatomical features such as an anteriorly sculpted sternum (often associated with tracheal coiling inside keel) in which the furcular process is fused to the anteroventral tip of the keel. Three extant gruine genera are recognized: Grus (10 species), Anthropoides (2 species), and Bugeranus (1 species). These genera are defined on the basis of soft anatomical features, although their monophyly has not been addressed by phylogenetic analysis. Fossil balearicines are known from the lower Eocene and later deposits in Eurasia, whereas Gruines date from the late Miocene (Brodkorb 1967). Evolutionary relationships among cranes have been addressed with a variety of different approaches during the past two decades. Archibald (1976) derived the species groups shown in Table 1 on the basis of similarities in unison

DNA/DNA hybridization studies of the carnivorous marsupials. I: The intergeneric relationships of bandicoots (Marsupialia: Perameloidea)

SummaryA complete suite of comparisons among six bandicoot species and one outgroup marsupial was generated using the hydroxyapatite chromatography method of DNA/DNA hybridization; heterologous comparisons were also made with three other bandicoot taxa. Matrices of ΔTms, Δmodes, and ΔT50Hs were generated and corrected for nonreciprocity, homoplasy, and, in the case of ΔTms, normalized percent hybridization; these matrices were analyzed using the FITCH algorithm in Felsensteins PHYLIP (version 3.1). Uncorrected and nonreciprocity-corrected matrices were also jackknifed and analyzed with FITCH to test for consistency. Finally, sample scores for ΔTm, Δmode, and ΔT50H matrices were bootstrapped and then subjected to phylogenetic analysis. These manipulations were carried out, in part, to address criticisms of the statistics used to summarize DNA/DNA hybridization (especially T50H) and the method itself. However, with the exception of an unresolved trichotomy among the twoEchymipera species andPeroryctes longicauda, all trees showed the same branchpoints. Except in the case of the tree generated from reciprocal-corrected ΔTm data, nodes were stable under jackknifing; and, again excepting the above-mentioned trichotomy, all nodes were supported by 95% or more of the bootstrapped trees. These results suggest that, despite arguments to the contrary, all three summary statistics can be valid for DNA/DNA hybridization data. Of taxonomic interest is the placement ofEchymipera spp. andPeroryctes longicauda together and separate from the more distantPeroryctes raffrayanus; the genusPeroryctes is thus at least paraphyletic. The trees further groupedEchymipera-plus-Peroryctes as the sister group ofIsoodon-plus-Perameles. Limited hybridizations withMacrotis lagotis suggest that its current position as representative of an entirely distinct family of perameloids is correct.

PHYLOGENETIC MEASURES OF BIODIVERSITY: A COMPARISON AND CRITIQUE

Abstract Several recent authors have proposed quantitative measures of taxonomic diversity that differ from traditional species-richness and abundance indices. These new methods emphasize phylogenetic branching order and levels of character divergence (including genetic variation) among species. In this study, seven phylogenetic diversity measures are compared using a DNA hybridization data set for cranes. The measures agree in identifying the species that contribute the most and the least to group diversity, but disagree on ranks assigned to species at intermediate levels. Evaluation of alternative measures hinges on the relevance conservationists attach to character divergence beyond its use in reconstructing phylogeny. The use of any such measure is complicated by inconsistency between character sets and disagreement over the definitions of species and subspecies. Until some working consensus is reached on these issues, phylogenetic indices are unlikely to supersede more traditional measures of biodiversity. Substantial effort will be required to integrate phylogenetic diversity with other factors in conservation planning.

Mitochondrial phylogeography, subspecific taxonomy, and conservation genetics of sandhill cranes (Grus canadensis; Aves: Gruidae)

Six subspecies of sandhill cranes (Gruscanadensis) have been denoted based onperceived morphological and/or breedinglocality differences among them. Threesubspecies are migratory, breeding from thehigh arctic in North America and Siberia(lesser sandhill, G. c. canadensis),south through central Canada (Canadiansandhill, G. c. rowani) and into thenorthern United States (greater sandhill, G. c. tabida). A review of sandhill cranetaxonomy indicates that the size variation, onthe basis of which these subspecies were named,may be clinal and not diagnostic. The otherthree subspecies, all listed as endangered orthreatened, are non-migratory, resident inFlorida (G. c. pratensis), Mississippi(G. c. pulla), and Cuba (G. c.nesiotes). We used analysis of mitochondrialDNA control region (CR) sequences to determinewhether haplotypes representing currentsubspecies show any genetic cohesion or aremore consistent with a pattern of clinalvariation in morphology. CR sequences indicatethat only two highly divergent (5.3%) lineagesof sandhill cranes occur in North America: onelineage composed only of arctic-nesting G.c. canadensis, the other of the remainingNorth American subspecies (we lack data on theCuban population). The deep split betweenlineages is consistent with an estimatedisolation of approximately 1.5 Mya(mid-Pleistocene), while the distribution ofmutational changes within lineages isconsistent with an hypothesis of rapid,post-Pleistocene population expansions. Noother phylogeographic structuring is concordantwith subspecific boundaries, however, analysisof molecular variance indicates that there issignificant population genetic differentiationamong all subspecies except G. c. tabidaand G. c. rowani, which areindistinguishable. We suggest thatrecognition of the recently named G. c.rowani be abandoned.

Phylogenetic structure of the marsupial family dasyuridae based on cytochromeb DNA sequences

Archer provided the most recent and comprehensive suprageneric classification of dasyurid marsupials. Five extant subfamilies, two with constituent tribes, were recognized on the basis of morphological, serological, and allozyme data. Phylogenetic relationships among these groups, however, were totally unresolved. Subsequent molecular studies suggested that the endemic New Guinean subfamilies Muricinae and Phascolosoricinae are parts of larger Australian clades. Our objective in this study was to test the monophyly of Archers seven groups and estimate relationships among them using DNA sequences from the mitochondrial cytochromeb (cyt-b) gene. We report 657 bp ofcyt-b from 32 dasyuroid species. Phylogenetic analysis of these data leads to the following conclusions: (1) muricines form a clade within Phascogalinae that includes endemic New GuineanAntechinus species; (2) the two genera of Phascolosoricinae are part of a more inclusive Dasyurinae; (3) Sminthopsinae is monophyletic, but the tribes Sminthopsini and Planigalini are not; and (4) the dasyurine tribes Dasyurini and Parantechini are probably not monophyletic. Relationships among Sminthopsinae, Dasyurinae (including phascolosoricines), and Phascogalinae (including muricines) remain unresolved.

Systematics and Evolution of the Dasyurid Marsupial Genus Sminthopsis: II. The Murina Species Group

Genetic variation within the Murina species group, which includes S. murina, S. gilberti, S. leucopus, S. dolichura and S. archeri, was examined through analyses of complete 12S rRNA, partial control region mitochondrial DNA sequences and partial omega-globin nuclear DNA sequences. Sminthopsis butleri was found to be an additional member of the Murina group, and appears to be most closely related to S. leucopus rather than the morphologically similar S. archeri. This latter species appears to be the most divergent member of the group, and there is a possible sister relationship between S. murina and S. gilberti, as suggested by previous allozyme evidence. It appears that the systematic affinities of the taxonomically problematic northeastern Queensland populations of both S. murina and S. leucopus and a disjunct population of S. gilberti (from the Western Australia/South Australia border) are indeed with those respective species; although each appears to belong to a distinct morphological and genetic lineage. A specimen of S. leucopus from Queensland was found to be as divergent from each of the southeastern Australian S. leucopus subspecies as they are from each other, suggesting that this northern population of S. leucopus may also warrant recognition as a distinct taxon. Specimens of S. murina murina were found to be genetically divergent from each other, and this subspecies appears to be paraphyletic, as suggested by previous morphological evidence.

The evolution of protamine P1 genes in dasyurid marsupials

We report the complete DNA sequences of the protamine P1 gene and flanking regions for 13 species of the marsupial family Dasyuridae. The structure of the protamine locus is conserved in dasyurids and consists of two exons (of lengths 142–151 and 47 bp) separated by an intron (208–240 bp). A key feature of the dasyund intron is a 38–40 by duplication found in all species examined to date. This duplication apparently predates the radiation of modern dasyurid lineages and may be homologous to a similar feature in the marsupial mole (Notoryctes). Sequences from a species of Planigale demonstrate that this genus is unique among marsupials in possessing cysteine residues in its protamine P1 molecules. Cysteines may provide enhanced chemical stability for condensed sperm nuclei, a physiological feature that would converge on the common eutherian pattern. Phylogenetic analysis of the protamine genes yields a tree that is largely congruent with previous molecular systematic studies in two areas: (1) There are three main dasyurid lineages corresponding to the Sminthopsinae, Dasyurinae, and Phascogalinae; (2) Dasyurinae and Phascogalinae are sister groups. This study is the first estimate of dasyurid relationships based on a nuclear DNA sequence.

Complete Mitochondrial Genome Sequences and the Phylogeny of Cranes (Gruiformes: Gruidae)

ABSTRACT. We estimated phylogenetic relationships among all 15 extant species of cranes in the gruiform family Gruidae from complete sequences of their mitochondrial genomes. The gene order of crane mitochondrial genomes corresponds to that of the chicken and present few structural novelties compared with previously described birds. Sequences of the control region, particularly domains I and III, are highly divergent among species and include tandem repeats, duplications, and numerous indels. Phylogenetic analyses confirmed the well-established, reciprocal monophyly of clades Balearicinae (crowned cranes) and Gruinae (anatomically derived cranes), as well as previously identified lineages within Gruinae: Leucogeranus and the species groups Anthropoides, Canadensis, Antigone, and Americana. As in previous molecular phylogenies, Leucogeranus is resolved as sister to all other gruines. In addition, the Sandhill Crane (Grus canadensis) is resolved as sister to the Antigone species group, resulting in a Pacific Rim clade that has not previously been suggested. Only relationships among the Anthropoides, Americana, and Pacific Rim groups remain unresolved in our analyses. The crane fossil record provides reasonable calibration points for the most recent common ancestor of Gruinae (Middle Miocene) and the minimum age of Grus americana (Late Pliocene). Bayesian estimates of divergence dates from mitochondrial DNA sequences suggest that balearicines and gruines separated in the late Oligocene and that radiations of living species within these clades took place in the Neogene.